Genomic Epidemiology of Multi-Modal ESBL Gene Transmission among Enterobacterales in a Neonatal Unit

Antimicrobial resistance genes (ARGs) can spread via horizontal transfer or clonal expansion. We investigated the genomic epidemiology of extended-spectrum beta-lactamase (ESBL)--producing Klebsiella pneumoniae and Escherichia coli in a neonatal unit. Between January and November 2023, 53 ESBL isolates were obtained from 23 neonates via routine screening and clinical sampling. Long-read nanopore sequencing identified blaCTX-M-15 as the dominant ESBL gene, alongside blaCTX-M-65 and blaCTX-M-27. Among 49 blaCTX-M-15 isolates, 33 carried the gene on plasmids and in the remainder it was located on the chromosome. ESBL isolates belonged to one of seven MLST sequence types; E. coli isolates were dominated by ST131/ST131-like/ST5640 lineages, while K. pneumoniae were predominantly ST13. Plasmids (ESBL and non-ESBL-associated) clustered into 18 communities, five of which contained plasmid-bearing blaCTX-M genes. The largest cluster comprised IncFIB(K) plasmids from K. pneumoniae ST13, although these were predicted as "non-mobilizable" by MOB-suite and belonged to isolates from a clonally disseminated strain. No evidence of blaCTX-M dissemination via shared plasmids was identified. Meanwhile, chromosomal phylogenetic analysis identified four distinct clonal clusters with [≤]7 SNP differences (n = 10, 5, 5, and 2 patients). In this setting, genomic analysis supported clonal dissemination of several blaCTX-M-associated strains as the main outbreak mechanism, affecting 20/23 neonates, rather than plasmid-mediated transmission.